Double-stranded p-siRNA molecule and p-siRNA recombinant plasmid for suppressing sox2 gene expression and application thereof

A recombinant plasmid and gene expression technology, applied in the field of biomedicine, can solve the problems of reducing, difficult to develop small molecule drugs of SOX2 protein, off-target and so on

Active Publication Date: 2019-10-15
INNER MONGOLIA MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The spatial conformation of the SOX2 protein is relatively special, and it lacks a pocket domain that binds to small molecule compounds, making it difficult to develop small molecule drugs that inhibit the activity of the SOX2 protein
Although small interfering RNA targeting SOX2mRNA can inhibit the translation of SOX2, its off-target phenomenon is very serious, and it will reduce the expression of other members of the family, especially members that play a role in suppressing tumors

Method used

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  • Double-stranded p-siRNA molecule and p-siRNA recombinant plasmid for suppressing sox2 gene expression and application thereof
  • Double-stranded p-siRNA molecule and p-siRNA recombinant plasmid for suppressing sox2 gene expression and application thereof
  • Double-stranded p-siRNA molecule and p-siRNA recombinant plasmid for suppressing sox2 gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: p-siRNA inhibits SOX mRNA expression

[0030] Step 1, reagent and reagent preparation thereof, in the following examples, the used

[0031] Instruments: fluorescent quantitative PCR instrument (ABI 7500); PCR instrument (Biometra, T1 Thermobycler); cell culture box (Thermo), etc.

[0032] Materials and reagents: RNAiMate (genepharma), RPMI 1640 medium (Gibco), Trizol (Tiangen), reverse transcription kit (DRR014A PrimeScript TM , TaKaRa), SYBR Premix Ex Taq (TaKaRa) 24-well culture plate (Nunc), etc.

[0033] PCR primers:

[0034] SOX2 forward primer: 5'-TACAGCATGTCCTACTCGCAG-3'

[0035] SOX2 reverse primer: 5'-GAGGAAGAGGTAACCACAGGG-3'

[0036] GAPDH forward primer; 5'-TGTCCCCACTGCCAACGTGTCA-3'

[0037] GAPDH reverse primer: 5'-GCGTCAAAGGTGGAGGAGTGGGT-3'

[0038] Step 2. Chemical synthesis of p-siRNA

[0039] According to the sequence of 141bp to 118bp upstream of the transcription start site of SOX2 promoter, design p-siRNA:

[0040] Sense strand: 5'-...

Embodiment 2

[0048] Example 2: WesternBlot detection of p-siRNA inhibition of SOX2 protein expression

[0049] Human lung cancer cell H446, liver cancer cell HepG2, colon cancer cell HT29 and cervical cancer cell Hela were divided into 1.5×10 5 cells / well were seeded into 6-well cell culture plates. At 37°C, 5% CO 2 Culture the cells in the incubator for 24 hours, press siRNA-Mate TM For transfection according to the steps in the instructions, set up negative control group and 80nmol / L p-siRNA group. After 48 hours of transfection, aspirate the medium, wash twice with PBS, add 100μL cell lysate, and lyse on ice for 10min. The cells were scraped off with a cell scraper, transferred to a 1.5ml centrifuge tube, centrifuged at 10,000rpm for 5min, and the supernatant was taken to determine the protein concentration. Take 20 μg of protein from each group for SDS-PAGE, transfer to PVDF membrane after electrophoresis, block and incubate with SOX2 primary antibody and goat anti-rabbit secondary ...

Embodiment 3

[0051] Example 3: p-siRNA inhibits tumor cell proliferation

[0052] Human lung cancer cell H446, liver cancer cell HepG2, colon cancer cell HT29 and cervical cancer cell Hela were mixed with 5×10 3 cells / well were seeded into 96-well cell culture plates. At 37°C, 5% CO 2 Culture the cells in the incubator for 24 hours, press siRNA-Mate TM Transfect p-siRNA according to the steps in the instructions, set up negative control group, 20nmol / L p-siRNA group, 40nmol / L p-siRNA group, 80nmol / L p-siRNA group and 120nmol / L p-siRNA group, transfect 48 After 1 hour, add 20 μL MTT (5 mg / mL) to each well, continue to incubate at 37° C. for 4 h, remove the medium, add 200 μL DMSO, and measure the absorbance at 490 nm wavelength.

[0053] Result analysis: p-siRNA can significantly inhibit the proliferation of four tumor cells in a concentration-dependent manner ( image 3 ).

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Abstract

The invention discloses a double-stranded p-siRNA molecule and p-siRNA recombinant plasmid for inhibiting the SOX2 gene expression, and an application thereof. The p-siRNA is any one of the following double-stranded RNA molecules: sense strand represented as 5'-CCCCGCGCGGCCGGCGGCGCGGGA- 3', and antisense strand represented as 5'-UCCCGCGCCGCCGGCCGCGCGGGG- 3'; or sense strand represented as 5'-CCCCGCGCGGCCGGCGGCGCGGGAtt- 3', and antisense strand represented as 5'- UCCCGCGCCGCCGGCCGCGCGGGGtt - 3'. The invention provides a broad-spectrum double-stranded p-siRNA and p-siRNA recombinant plasmid of a novel, efficient and targeting SOX2 gene, and also discloses the application of the broad-spectrum double-stranded p-siRNA and p-siRNA recombinant plasmid in kits and preparing anti-tumor drugs.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a double-stranded p-siRNA molecule and p-siRNA recombinant plasmid for inhibiting the expression of SOX2 gene and application thereof. Background technique [0002] Malignant tumors have become a major killer that threatens people's health and lives. There are more than 10 million new cases and more than 2.5 million deaths in the country every year. One person in every 7-8 people dies of malignant tumors. At present, the main treatment for tumors is to use surgery to remove the lesion and to kill tumor cells through radiotherapy and chemotherapy. However, surgery is difficult to remove small disseminated lesions. Traditional radiotherapy and chemotherapy lack specificity. While killing tumor cells, they also cause severe trauma to normal cells in the body. Some chemotherapy drugs also promote tumor metastasis or turn normal tumor cells into cancer stem cells. Therefore, th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85A61K31/713A61P35/00
Inventor 贾永峰王振飞施琳云芬郑媞杨永雁冯立李颖
Owner INNER MONGOLIA MEDICAL UNIV
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